The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The relative simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general. We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome in living cells. Using similar DNA recognition systems of different specificities, we can now label up to three chromosomal loci simultaneously, using three differently colored fluorescent proteins. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. This effort has been greatly augmented by collaboration with the laboratory of Flemming Hansen, the Technical university of Denmark. With him, have developed automated methods for the measurement of the positions of fluorescent foci in the cells that permits accurate measurement of thousands of cells from microscopic images. We are also developing rapid methods for the analysis of the large data sets that we are able to collect. These methods provide us with powerful tools for the investigation of the replication and segregation dynamics of the chromosome. We have been able to disprove the currently popular model for chromosome segregation involving simultaneous segregation of the bulk of the DNA. Rather, we show clearly that DNA is segregated progressively as it is replicated. Our investigations are revealing unexpected features of DNA organization and motion, including the fact that the two arms of the circular chromosome lie in opposite halves of the resting cell. We have been able to conclude that DNA segregation proceeds in concert with replication in a process that may resemble the formation of separable sister chromatids in higher organisms. In the past year, we have made substantial progress toward understanding chromosome segregation at fast growth rates, where the initiation of chromosome replication becomes uncoupled from the cell division cycle and the cells become functional diploids. Under these conditions, cell division occurs while chromosome replication is ongoing. We have confirmed that segregation is driven directly by replication so that segregation of chromosome domains can occur in generations previous to the one in which the regions are placed in separate cells by cell division. Using multiple fluorescent markers around the chromosome and three-dimensional analyses of their locations, we have mapped the topology of the replicating chromosome and its development throughout the cell cycle. The nucleoid was essentially found to be a hollow tube with only the origin-proximal region occupying its core. The mechanism places the origins of replication in segregation zones near the cell radial axis;one at the cell center and two near the outer ends of the nucleoid mass. The daughter markers are then actively separated along the cell long axis. As the replication forks progress away from the origin, subsequent paired markers are drawn into the segregation zone and the individual copies separate in turn in a symmetrical fashion. The two forks emerging from each origin operate together, and the two chromosome arms are intermixed. Origin-proximal markers segregate from the segregation zones first, and segregation is progressive. Thus each cell quarter shows a tendency to have the markers ordered into map order, but with both chromosome arms superimposed. Segregation of late markers occurs from the cell center and segregation of the earlier markers occurs from the nucleoid boarders, adding to the length of the nucleoid tube. At the nucleoid boarders, the origin-proximal markers add to the length of the sparsely occupied core whereas distal arm markers are added to the outer shell, near the membrane surface. Thus, origin regions are always near the radial axis and distal arms are at the nucleoid surface. The visible properties of DNA replication and segregation need to be linked to the biochemical and structural properties of the macromolecules involved in the key events. We have made significant progress in understanding the role of the SeqA protein that has been implicated in both replication and segregation of the chromosome. In collaboration with Dr. Alba Guarne (McMaster University) we have solved the crystal structure of the entire SeqA protein in a complex with its cognate DNA sequence. Using the structure as a guide, we have constructed mutant proteins and have determined their effects on DNA replication and segregation. In contrast to several extant publications, we have recently shown that SeqA protein at the replication forks is not required for proper chromosome segregation. Although it plays an essential role in governing origin replication initiation, we find that it is not directly involved in origin segregation either. Rather, SeqA appears to be directly involved in the detection of DNA lesions and their repair via the mismatch repair system. Several of the key elements in the mismatch repair system are conserved from bacteria to humans and defects are responsible e for several human cancer types. Specifically, we found that the SeqA protein co-localizes with SeqA at base-pair mismatches that are produced by inaccurate DNA replication at the replication forks. The extent of the involvement of SeqA protein in mismatch recognition and repair will be a major thrust of our research in the coming year.